国家神经系统疾病临床医学研究中心数据管理与共享系统

发表杂志：Nature neuroscience

发表类型: Article

影响因子(IF): 24.884

PMID: 33257875 Pubmed原文链接 全文下载

Normal aging is characterized by a gradual loss of various physiological functions across all organ systems within the body. During this process, one of the least understood aspects of normal aging is the coordination of its stereotyped progression in all organ systems. The immune and nervous systems are two unique body systems that co-evolve during aging and simultaneously exert broad control over all organs. Although alterations in the systemic control of immune responses in the periphery during aging of the nervous system are evident1,2,3, how aging of the nervous system affects immune responses within the brain and whether these changes affect the normal brain aging process remain elusive. The augmented basal inflammatory levels in the periphery that develop with advanced aging have long been known as ‘inflammaging’, which suggests that the immune system participates in the aging process of all organ systems4,5,6. In the periphery, aging induces inflammasome activation and production of interleukin-1β (IL-1β) and tumor necrosis factor that significantly correlate with cardiovascular risks, metabolic disturbances and macular degeneration7,8,9. In normal brain aging, the increased inflammatory activity is characterized by glia activation accompanied by accumulation of hematogenous lymphocytes and myeloid cells10,11. Evidence suggests that genetic deficiency of T lymphocytes results in cognitive deficits in young animals and that T lymphocyte infiltration into the aged subventricular zone (SVZ) of the brain is associated with a decline in neurogenesis12,13,14, thereby implying that lymphocytes might influence brain function during the aging process. As aging drives cognitive and regenerative impairments in the adult brain that cause increased susceptibility to neurological diseases6,15, it is critical to gain mechanistic insights into whether and how the immune system, especially its cellular compartment, participates in normal brain aging. In this regard, a number of questions need to be resolved. First, since the exact inflammatory signals during normal brain aging are unknown, what are the signals that maintain immune cells in the aged brain? Second, are immune cell subsets universally recruited and maintained in the aged brain or does each subset behave differently? Third, do immune cells exhibit different phenotypes and functions after homing to the brain, and why? Fourth, where do immune cells reside in the aged brain and what are their effector functions? Finally, most prior studies of immune cells in the aged brain have been performed with rodents, and the relevance of these findings to human aging is unclear. The present study was carried out to address these questions.

Postmortem human brain tissues Human brain sections were provided by Shanxi Medical University (Shanxi, China). All brain tissues were acquired during sudden unexpected death without brain injury or prior neurological diseases before death. Among the total of 23 samples studied, 10 were from young individuals and 13 were from aged individuals (young: 30.5 ± 2.6 years; aged: 71.6 ± 1.5 years, mean ± s.e.m.; P < 0.01, unpaired t-test). Brain tissues were collected within 4 h after death. Postmortem histopathological examination of brain sections by a neuropathologist confirmed that no pathological alterations were related to neurological or neuropsychiatric diseases. Human blood samples Human blood samples were obtained from Beijing Tiantan Hospital. The young group included 19 participants and the aged group included 17 participants (young: 29.8 ± 1.7 years; aged: 73.5 ± 1.6 years, mean ± s.e.m.; P < 0.01, unpaired t-test). Individuals were chosen on the basis of standardized inclusion and exclusion criteria as follows. Inclusion criteria: (1) individuals met the age cutoffs for different groups; (2) general health, body mass index between 18 and 34 and normal basic laboratory tests; (3) memory performance on logical memory within normal limits, and neurological exam is normal. Exclusion criteria: (1) neurological disorders, psychiatric disorders or active/uncontrolled depression; (2) cancer in the past 12 months; (3) drug or alcohol abuse in the past 2 years; (4) anti-Parkinson’s disease medications, CNS stimulants, antiepileptic drugs, cortisone or insulin treatment. The inclusion of human participants and supporting documentation were approved by the Beijing Tiantan Hospital Ethics Committee (KY2019-050-02). Informed consent was obtained from all participants or a legally acceptable surrogate at the time of enrollment. Mice All animal experiments were performed in accordance with the Animal Research: Reporting in vivo Experiments guidelines. All animal experiments were performed in accordance with the approved protocols by the Animal Care and Use Committees of Beijing Tiantan Hospital and Tianjin Medical University General Hospital (201902026). All studies were performed using adult male mice at the indicated ages. C57BL/6, Cd1d−/−, B6.Cg-Gt (ROSA) 26S, FGD5–CreERT2, Nestin–CreERT2 and DCX–mRFP mice were purchased from The Jackson Laboratory. FGD5–CreER–tdTomato mice were obtained by crossing FGD5–CreERT2 mice with B6.Cg-Gt (ROSA) 26S mice as previously described50. Nestin–CreER–tdTomato mice were obtained by crossing Nestin–CreERT2 mice and B6.Cg-Gt (ROSA) 26S mice. Rag2−/− and Rag2−/−γc−/− mice were purchased from Taconic. NK1.1–tdTomato mice were generated as previously described51. All mutant mice were backcrossed to the B6 background for at least 12 generations. All mice were maintained under pathogen-free conditions and housed with no more than five animals per cage under standardized light–dark cycle conditions with ad libitum access to food and water. The vivarium was maintained under controlled temperature (21 ± 1 °C) and humidity (50–60%), with a 12-h diurnal cycle (lights on: 7:00–19:00). Flow cytometry Human peripheral blood mononuclear cells were isolated from whole blood by centrifugation using a human lymphocyte isolation kit (Solarbio Life Science). Peripheral blood mononuclear cells were then stained with fluorochrome-conjugated antibodies to evaluate NK cell counts and phenotype51,52,53. Mice were killed by lethal anesthesia via isoflurane, and after perfusion with cold PBS, brain, spleen, liver, lung and thymus were removed. Brain or microdissected dentate gyrus tissues were minced and incubated with collagenase IV and DNase at 37 °C for 30 min. After removing the myelin debris by centrifugation in 30% Percoll, single cells were suspended in 1% BSA followed by antibody staining. Spleen, liver, lung and thymus tissues were minced and strained through a 70-µm cell strainer, and red blood cells were removed using lysis buffer. Thereafter, single cells were suspended in 1% BSA, followed by antibody staining. In experiments using blood and bone marrow, single cells were collected after removal of red blood cells using lysis buffer. Single-cell suspensions were placed in conical centrifuge tubes (106 cells per tube), and then stained with flow cytometry antibodies. For intracellular staining, cells were fixed in fixation buffer for 20 min after surface marker staining. After washing twice in permeabilization buffer, cells were incubated with antibodies in the staining buffer for 45 min. The following antibodies were used: CD3 (HIT3a, 555342; 17A2, 557869, BD Biosciences); CD56 (B159, 561905, BD Biosciences); CD69 (L78, 341652, BD Biosciences); CD25 (M-A251, 560503, BD Biosciences); granzyme B (clone GB11, 560213, BD Biosciences; NGZB, 25-8898-82, eBioscience); CD45 (30-F11, 565478, BD Biosciences); CD11b (M1/70, 101263, BioLegend); NK-1.1 (PK136, 551114, 552878, BD Biosciences); CD4 (clone RM4-5, 552775, BD Biosciences); CD8a (53-6.7, 557654, BD Biosciences); Ly-6G (1A8, 127614, BioLegend); B220 (RA3-6B2, 553093, BD Biosciences); IL-27 (IC1834R, 234205, R&D Systems); perforin (eBioOMAK-D, 12-9392-82, eBioscience); ULBP-1/MULT-1 (BaF3, FAB2588V, R&D Systems); CD1d (1B1, 553846, BD Biosciences); RAE1-γ (clone CX1, 130110, BioLegend); and Qa-1 (6A8.6F10.1A6, 744387, BD Biosciences). Data were collected on a FACSAria III flow cytometer, and analyzed using FlowJo software. For cell sorting, mouse dentate gyrus tissues were dissected and dissociated as described above. Single cells were incubated for 45 min with antibodies against the following cell surface markers: CD24 (M1/69, BD Pharmingen); CD15 (HI98, 17-0291-82, Thermo Fisher Scientific); CD29 (HMb1-1, 17-0291-82, eBioscience); GLAST (ACSA-1, Miltenyi Biotec); EGFR (E-35351, Invitrogen); CD45 (30-F11, 565478, BD Biosciences); and CD11b (M1/70, 101263, BioLegend). Different cell populations were isolated in a single sort using a BD FACS Aria with a 13 p.s.i. pressure and 100-μm nozzle aperture. Cell sorting was conducted using a FACSAria III instrument as previously described51,52,53. Cell cycle distribution was measured by flow cytometry using a cell cycle assay kit (ab112116, Abcam). Spatial cognition evaluation The Morris water maze was used to evaluate the cognitive function of aged mice. The test was conducted in two periods; that is, the training test and the probe test. During the training period, mice were introduced to a circular pool, in which a hidden round platform submerged in the water was placed in the northeastern quadrant, and mice could escape by finding this platform. In each trial, mice were introduced to the pool through the west side and allowed to swim at a maximum of 120 s to find the platform. The trial was ended after mice found the platform or swam for 120 s without finding the platform. Mice that successfully found the platform within 120 s were allowed to stay on the platform for 10 s, and mice that failed to locate the platform were placed on the platform by the experimenter and were also allowed to stay for 10 s. The training was then repeated by introducing the mice to the pool through the south side. These two trials were considered as one complete session, and two sessions were performed per day for 4 days. The swimming velocity, escape latency and total distance swam during a trial were recorded. The water temperature in the pool was maintained at 24 °C during the test. After the training period, mice were subjected to the probe test on the fifth day (in which the platform was removed), and a single trial was conducted by introducing the mice to the pool through the southwestern quadrant. Each mouse was allowed to swim for 120 s, during which the time spent in the northeastern quadrant and crossing times of the target platform area were recorded. EthoVision 3.1 tracking system (Noldus Information Technology) was used for data acquisition. Electrophysiology After euthanasia, mouse brain tissues were incubated in artificial cerebrospinal fluid (ACSF; containing (in mM): 119 NaCl; 2.5 KCl; 26 NaHCO3; 1.3 MgSO2; 1.0 NaH2PO4; 2.5 CaCl2; and 11 glucose). The ACSF was continuously bubbled with 95% O2–5% CO2 (carbogen). The brain slices containing the dentate gyrus were prepared with a vibratome (Vibratome) at a thickness of 400 μm, and then incubated in ACSF at room temperature for at least 1 h before recording. Thereafter, brain slices were transferred to a liquid–air interface chamber (Warner Instrument) and suspended on a nylon net in a bath of continuously dripping oxygenated ACSF (2.0–2.5 ml min−1). Humidified carbogen was passed along the upper surface of the slices, and the temperature was maintained by a feedback circuit to 31 ± 0.5 °C. Standard field potentials were recorded in the hippocampal dentate gyrus region. A stimulating electrode was placed in the angular bundle of the perforant path and a recording electrode in the hilus of the dentate gyrus, and stimulation was delivered using a Model 2100 A-M Systems Isolated Pulse Stimulator. A stimulus intensity that induced half maximal intensity in the input–output relationship was used. The baseline synaptic potentials were first recorded for 20 min, then a theta-burst tetanic stimulation (15 burst trains at 5 Hz, each train containing five pulses at 100 Hz) was delivered to induce LTP. The baseline intensity-evoked field excitatory postsynaptic potentials (fEPSPs) were recorded for 60 min with 0.33 Hz. All evoked responses were recorded by an Axoclamp-2B amplifier. Data were acquired using pClamp 10.2 software (Molecular Devices). Parabiosis Parabiosis was conducted of aged wild-type mice and NK1.1–tdTomato mice, which were similar in weight and size. Mice were placed in the same cage for 2 weeks before parabiosis surgery to ensure harmonious cohabitation. One mouse of each strain was placed in one cage. During surgery, mice were anesthetized by inhalation of 3.5% isoflurane and maintained by inhalation of 1.0–2.0% isoflurane in 70% N2O and 30% O2. A skin incision was made in the body from the elbow to the knee. The elbow joints of two mice were connected by a 3-0 nonabsorbable suture, then the same process was conducted at the knee joints. Then, the side skin of the two mice was connected by a 5-0 absorbable Vicryl suture. Buprenorphine SR was given (1 mg per kg) subcutaneously for analgesia immediately after surgery and repeated 3 days thereafter. Mice were connected for 2 weeks to allow efficient exchange of blood circulation, then the paired mice were separated. Four weeks after separation, the tdTomato+ NK cell percentage in the total NK cell population was measured by flow cytometry. Tamoxifen treatment Tamoxifen was dissolved in ethanol/corn oil (1:9) at a concentration of 30 mg ml−1, and mice were intraperitoneally injected with 100 µl (3 mg per mouse) daily for 5 days. Four weeks later, the bone marrow, spleen, blood and brain tissues were obtained to measure tdTomato+ NK cells. Aged Nestin–CreER–tdTomato mice were treated with the anti-NK1.1 mAb every 5 days until the end of experiments. At the second day of the first anti-NK1.1 mAb administration, tamoxifen was given to the mice for 5 days. Mouse dentate gyrus tissues were collected at 4 weeks after the first anti-NK1.1 mAb treatment. Single-cell RNA sequencing CD45high single cells were sorted by flow cytometry for single-cell RNA sequencing. A 10× Genomics Chromium single-cell 3′ v.2 kit and its protocol were used to prepare the libraries. HalioDx performed the RNA sequencing on a NextSeq500 machine with a sequencing depth of at least 50,000 reads per cell. Single-cell RNA-sequencing data processing Raw FASTQ files were processed with Cell Ranger software (v.1.3.1), which performs alignment, filtering, barcode counting and unique molecular identifier (UMI) counting. Reads were first simultaneously aligned with the Grch38 genomes using CellRanger software (v.1.3.1) for their assignment as murine sequences. Low-quality cells were excluded in an initial quality-control (QC) step by removing genes expressed in fewer than three cells, cells with fewer than 200 genes expressed and cells expressing more than 2,500 genes. Cells with more than 0.05% of mitochondrial-associated genes among their expressed genes were also removed. Library-size normalization was performed on the UMI-collapsed gene expression values for each cell barcode by scaling by the total number of transcripts and multiplying by 10,000. The data were then log-transformed before further downstream analysis. R studio platform was used for downstream analysis with the Seurat package. We first selected genes with a high variance by using the FindVariableGenes function, then reduced the dimensionality of our data by principle component analysis (PCA) and identified by random sampling 20 significant principal components (PCs) for each sample with the PCElbowPlot function. Cells were clustered with Seurat’s FindClusters function. For visualization, we applied RunTsne to the cell loadings of the previously selected PCs with default perplexity to view the cells in two dimensions. Marker genes were defined as genes with an adjusted P value of <0.05 tested with a nonparametric Wilcoxon rank-sum test. Analysis of variance (ANOVA) was performed using the R function aov to identify differentially expressed genes, and the difference between each cluster pair was tested using Tukey’s range test implemented in R function TukeyHSD. Then, differentially expressed genes of each cluster were identified as those meeting the following criteria: (1) false discovery rate (FDR)-adjusted P value of F test of <0.05; (2) P values of the Tukey’s range test comparing the cluster under consideration and others <0.05. Marker genes were defined as genes with an adjusted P value of <0.05 tested with a nonparametric Wilcoxon rank-sum test. PCA was performed on the mean expression of all genes between clusters. PCA gene loadings for PC1 and PC2 corresponded to the genes making the largest contributions; that is, accounting for 40% of the total information for each PC. Heatmaps were generated from the scaled expression (log TPM) values for the discriminating gene sets defining each subset, with adjusted P values of <0.05 with a nonparametric Wilcoxon rank-sum test. The color scale is based on the z-score distribution, from −2 (purple) to 2 (yellow). For Gene Ontology (GO) enrichment analysis, enrichment scores (P values) for selected numbers of GO annotations were calculated using a hypergeometrical statistical test with a threshold of 0.05. The data were plotted as −log10 of the P value after Benjamini and Hochberg correction. For mouse data, enrichment was calculated for the union of the marker genes across samples. The background was the union of all genes expressed in mouse samples. The background was all the genes expressed within individual samples. Samples were ranked according to the lowest P value of the leader subsets for each GO annotation. RNA sequencing Total RNA extraction Total RNA was extracted from the tissues using TRIzol (Invitrogen) according to the instruction manual. About 60 mg of tissues was ground in liquid nitrogen in a 2-ml tube. After homogenization for 2 min, samples were rested horizontally for 5 min. The mix was centrifuged for 5 min at 12,000 × g at 4 °C, and then the supernatant was transferred into a new tube with 0.3 ml chloroform/isoamyl alcohol (24:1). The mix was vigorously shaken for 15 s, and then centrifuged at 12,000 × g for 10 min at 4 °C. After centrifugation, the upper aqueous phase was transferred into a new tube and an equal volume of isopropyl alcohol was added, then centrifuged at 13,600 r.p.m. for 20 min at 4 °C. After removing the supernatant, the RNA pellet was washed twice with 1 ml 75% ethanol, and then the mix was centrifuged at 13,600 r.p.m. for 3 min at 4 °C to collect residual ethanol, followed by air drying for 5–10 min. Subsequently, RNA was resuspended in 25–100 μl DEPC water, and then qualified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific). mRNA library construction Oligo (dT)-attached magnetic beads were used to purify mRNA. Following purification, the mRNA was fragmented into small pieces using divalent cations at an appropriate temperature. Then first-strand complementary DNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis step. Afterwards, A-Tailing Mix and RNA Index adapters were added and incubated to end repair. The cDNA fragments obtained from the previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on an Agilent Technologies 2100 Bioanalyzer for QC. The double-stranded PCR products from the previous step were heated, denatured and circularized by the splint oligo sequence to obtain the final library. The single-strand circle DNA was formatted as the final library. The final library was amplified with phi29 to make DNA nanoballs, which had more than 300 copies of one molecular species. The DNA nanoballs were loaded into the patterned nanoarray, and single-end 50 base reads were generated on a BGIseq500 platform (BGI-Shenzhen). Quantitative real-time PCR Quantitative real-time PCR was performed for gene expression analysis. Total RNA was extracted using column purification (Qiagen). First-strand cDNA was generated with random primers using iScript cDNA synthesis kits (Bio-Rad). Analysis was performed on a Bio-Rad Cycler system (Bio-Rad) using the following conditions: initial denaturation for 10 min at 95 °C; 40 cycles of 15 s denaturation at 95 °C; 30 s annealing at 60 °C; 1 min extension at 72 °C. Analysis was performed according to the ΔΔCt method using GAPDH as a housekeeping gene. RNA-sequencing data processing The fragments per kilo base of exon per million fragments mapped method was used to calculate gene expression levels. The clustering analysis of gene expression of all the samples in random sequence was performed with Cluster (v.3.0) and Java Treeview software. Heatmaps were generated using the R heatmap package. Differentially expressed genes (DEGs) were defined as log2-ratio ≥ 1 and q value (corrected P value) ≤ 0.001. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations were conducted at BGI Genomics. Briefly, gene numbers were computed for every term, and the differentially expressed genes (DEGs) were subjected to a hypergeometric test. Significantly enriched GO terms were determined based on a q value of ≤0.05 after FDR correction. The formula used to calculate the q values to determine significantly enriched KEGG pathways was the same as for GO analyses. For prediction of the transcription factor families encoded by the DEGs, the open reading frame of each DEG was detected using the Getorf software (http://emboss.sourceforge.net/apps/cvs/emboss/apps/getorf.html), and then mapped to the Animal TFDB databases with the Diamond software. Data mining and figure presentation process, including the GO term analysis, the gene–gene interaction analysis, heatmap and clustering were performed using the Dr. Tom BGI in-house customized data-mining system. RNA silencing Single-cell suspensions were prepared from dissected dentate gyrus of young (3 months old) and aged (18 months old) DCX–mRFP reporter mice. RFP+ neuroblasts were then sorted using flow cytometry. Short interfering RNA (siRNA) transfections were performed for mTOR and Il27 gene knockdown using Lipofectamine 2000 (Life Technologies). The mTOR siRNA or Il27 siRNA plasmids were from Santa Cruz (sc-35410, sc-72185, Santa Cruz). BrdU labeling For BrdU labeling, mice were intraperitoneally injected with 150 mg per kg BrdU twice per day for 3 days and killed 24 h after the last injection. Brains were perfusion-fixed with 4% paraformaldehyde, followed by overnight post-fixation in the same fixative at 4 °C. Sections (30 μm) were prepared the next day using a Vibratome (Thermo Scientific, HM550). After three rinses in PBS, sections were incubated in 2 N HCl for 30 min (for BrdU staining). The sections were then rinsed, preincubated with 5% blocking serum in 0.3% Triton X-100 for 1 h, then overnight with primary antibodies BrdU (1:200, ab6326, Abcam) at 4 °C, followed by a 1 h incubation at room temperature with Alexa Fluor-conjugated secondary antibodies (Invitrogen). Immunostaining Immunostaining of brain sections was performed as previously described51,52,53. The primary antibodies were incubated at vendor-suggested concentrations at 4 °C overnight. After washing with PBS for five times, tissue sections were then incubated with Alexa Fluor-conjugated secondary antibodies at 4 °C overnight. After washing with PBS for five times, the nuclei were stained by a mount medium containing 4′,6-diamidino-2-phenylindole (DAPI). The following antibodies were used: NKP46 (1:250, 195314, MAB1850, R&D Systems; 1:250, AF2225, R&D Systems); CD3 (SP162, 1:100, ab135372, Abcam); CD19 (6D5, 1:250, ab25232, Abcam); CD14 (SP192, 1:100, ab230903, Abcam); CD66b (1:200, ab214175, Abcam); CD69 (EPR21814, 1:500, ab233396, Abcam); granzyme B (EPR22645-206, 1:200, ab255598, Abcam); CD31 (MEC 13.3, 1:10, 553370, BD Biosciences; ab32457, 1:100, Abcam); DCX (1:200, ab18723, Abcam); BrdU (1:200, ab6326, Abcam); nestin (1:500, ab6142, Abcam); caspase-3 (1:500, ab13847, Abcam); Ki67 (1:200, SolA15, 12-5698-82, Thermo Fisher Scientific); and IL-27 (1:200, 516912, BioLegend). Images were captured using a Zeiss confocal microscope and analyzed with ImageJ software. Western blotting The neuroblasts were sorted from young (3 months old) and aged (18 months old) mice by flow cytometry for western blotting. Isolated cells were lysed in RIPA buffer (Solarbio) with 1 mmol per liter phenylmethanesulfonyl fluoride (Solarbio). After centrifugation, the supernatants were collected as total proteins. Then the proteins were separated by 10% SDS–PAGE and transferred onto a nitrocellulose membrane (Amersham Biosciences). Immunoblot analysis was performed with primary antibodies against P16 (1:500, sc-377412, Santa Cruz), P21 (1:500, sc-6246, Santa Cruz) and P53 (1:500, sc-126, Santa Cruz) at 4 °C overnight, and β-tubulin (2146, Cell Signaling Technology) was used as internal control. Then, the membrane was incubated with horseradish-peroxidase-conjugated anti-rabbit or anti-mouse secondary antibody (ZSGB-BIO) for 1 h at room temperature. The intensity of chemiluminescence was measured using an ImageQuant LAS 4000 apparatus (GE Healthcare Life Sciences). Cellular senescence detection A green florescence senescence detection kit (C10840, Invitrogen) was used to determine β-galactosidase expression. The staining process followed the vendor’s manual. In brief, young and aged mice were killed, and single-cell suspensions of dentate gyrus were collected. After staining with cell surface markers, cell suspensions were incubated with working solution at 37 °C for 2 h without CO2. After washing in PBS, cells were measured by flow cytometer or visualized under a microscope to capture images. mAb treatment To deplete NK cells in vivo, we used a mouse anti-NK1.1 mAB (PK136, American Type Culture Collection). Mouse IgG2a (Sigma-Aldrich) was used as the control. Aged mice received anti-NK1.1 mAb or IgG control at a dosage of 100 µg per mouse every 5 days until the end of experiments. Depletion of NK1.1+ cells was confirmed by flow cytometry and was consistently >90%. For in vitro neutralization of IL-27, an anti-IL-27 mAb or IgG control was added to the culture medium at a concentration of 10 µg ml–1. For in vitro inhibition of the RAE1–NKG2D pathway, an anti-RAE1 mAb (CX1, 130107, BioLegend) (20 µg ml–1) or IgG control was added into the NK–neuroblast co-culture. Microdialysis and proteome profiler array Microdialysis was performed to obtain the interstitial fluid from the dentate gyrus of young and aged mice. Mice were placed on a stereotaxic apparatus after anesthesia, a guide cannula was inserted into the dorsal dentate gyrus with the following coordinates: −2.3 mm anterior–posterior, −1.5 mm medial–lateral and −1.7 mm dorsal–ventral. The cannula was then fixed to the skull by dental cement. Three days after cannula implantation, a microdialysis probe filled with perfusion buffer (0.15% BSA–ACSF buffer, containing the following (in mM): 1.3 CaCl2, 1.2 MgSO4, 3 KCl, 0.4 KH2PO4, 25 NaHCO3 and 122 NaCl, pH 7.35) was inserted into the brain through the cannula. Mice were allowed to move in a free-moving chamber that avoids tangling of the tubes. Dialysis was performed for 3 h daily and repeated for three consecutive days. The collected interstitial fluid was used to measure soluble proteins by a mouse XL cytokine array kit (R&D Systems). Cell cytotoxicity assay Cell cytotoxicity was measured using a Live/Dead cell-mediated cytotoxicity kit (Molecular Probes, Thermo Fisher Scientific). Sorted neuroblasts were used as target cells and labeled with 3,3-dioctadecyloxacarbocyanine (DiOC18(3)), a green fluorescent membrane stain. Sorted neuroblasts were plated at a density of 5 × 105 cells per well in a 24-well plate. Thereafter, neuroblasts were treated with an anti-IL-27 mAb (10 µg ml−1) or the mTOR inhibitor rapamycin (1 µM) for 48 h. NK cells were sorted from the spleen and added at a ratio of 1:0.5–1:10 per well. The co-culture system was treated with an anti-RAE1 mAb (10 µg ml−1) or IgG for 24 h. Then, the membrane-impermeant nucleic acid counterstain propidium iodide (PI) was added. Live target cells (DiO+PI–), membrane-compromised target cells (DiO+PI+), live effector cells (DiO–PI–) and membrane-compromised effector cells (DiO–PI+) were quantified by flow cytometry. Quantification and statistical analyses All statistical details are provided in the figure legends, including statistical tests used, P values, n numbers, degrees of freedom (d.f.) and t or F values where relevant. No statistical methods were used to predetermine sample sizes, but our sample sizes were similar to those reported in previous publications51,52,53. Animals were randomly assigned to experimental groups. Randomization was based on the random number generator function in Microsoft Excel software. In experiments requiring tamoxifen injection, mice were excluded if they died after tamoxifen administration. For the in vitro experiments using siRNA, data points from cultured cells were excluded from analysis if there were no effect of knockdown. No data were excluded for other reasons. All results were analyzed by investigators blinded to experimental groups. Data distribution was assumed to be normal, but this was not formally tested. Statistical analysis was conducted on data from three or more biologically independent experimental replicates. Data are presented as the mean ± s.e.m. Statistical significance was determined by two-tailed unpaired Student’s t-test for two groups, ANOVA followed by Tukey’s post-hoc test for three or more groups or two-way ANOVA accompanied by Bonferroni post-hoc test for multiple comparisons. Values of P < 0.05 were considered significant. All statistical analyses were performed using Prism 7.0 software (GraphPad).